Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by a progressive impairment of cognitive functions and memory loss. Neurofibrillary tangles, β-plaques, synapse loss and neuron loss are major pathologic hallmarks of the brain in individuals with AD. A role for chronic inflammation in the brain degeneration of AD sufferers has been suggested since the neurodegenerative processes are accompanied by reactive astrogliosis and microglia activation (Dickson et al.; Rogers et al.). Immunopathological studies have shown that amyloid plaques are associated with clusters of activated microglial cells expressing pro-inflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6) and other inflammation-related regulatory molecules. The immune system and the central nervous system (CNS) are complex tissues and few studies have attempted to investigate their interaction. The CNS has been labelled an immunologically privileged site and the presence of the blood brain barrier (BBB) reinforced the view that immunosurveillance is absent within the CNS. However, recent CNS investigations point to the localised production of molecules with an immune function and its accessibility to a small number of lymphocytes and monocytes (Akiyama et al.; Rebenko-Moll et al.). Mechanisms of microglia recruitment into normal brain under physiological conditions have not been established. In animal models, a limited number of mononuclear phagocytes are continuously recruited into the disease-free brain where they either differentiate into microglia or remain a distinct population; studies of the diseased brain suggest a similar mechanism, but the process is accelerated. In an AD mouse model, a specific chemokine receptor facilitated the recruitment of microglia from within the brain and monocytes from the blood surrounding (β-amyloid deposits. Disrupting this process accelerated the disease and the mice rapidly died. These results suggest that microglia accumulation result in increase (β-amyloid deposition, particularly in and around blood vessels. Previous studies on gene polymorphism association with AD reinforced the view that altered immune responses could play a role in pathogenesis of AD (Chiapelli et al.). Moreover, several cytokines such as IL-1α, IL-1β, IL-2, IL-8, Interferon-γ (INF-γ) and tumour necrosis factor-a (TNF-a) have been found to be associated with senile plaques, to be secreted by activated microglia, and to be implicated in the development of neuritic plaques. Other molecules such as monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, P-selectin and L-selectin are involved in the trafficking of monocytes across the BBB.
Clinical diagnosis of AD often occurs long after the onset of the disease. It is usually first noticed by immediate family members who detect problems with short-term memory and unusual behaviour. Confirmation is achieved post-mortem by detecting the presence of the pathological hallmarks of the disease, amyloid β plaques and neurofibrillary tangles.
Current methodology to diagnose AD uses differential diagnosis which can involve patient interview and examination, details of patient's family and social history, blood testing to rule out other forms of dementia, psychological tests and neuroimaging. In addition, the presence of elevated levels of β-amyloid and tau proteins have been shown in many studies to be indicative of AD, but despite there being commercially available tests for these proteins, they are not yet clinically recognised for use in Alzheimer's diagnosis. The principal neuropsychological tests incorporate a memory test for diagnosing AD e.g. the Mini Mental State Examination (MMSE). Unfortunately, mild memory deterioration is commonly associated with ageing (as well as stress and depression) and often does not necessarily imply AD (Small et al.). Thus, there is great interest in both clinical and preclinical biomarkers.
Protein diagnostic biomarkers are an aid in the diagnosis of diseases. High specificity protein diagnostic markers of AD would provide an objective measure of disease progression and reduce costs. Such biomarkers are especially important for detecting the disease early (and discriminating AD from age-related mild memory deterioration) when therapeutic compounds have the greatest potential effect, but more importantly so that the patient and family can formulate a plan to manage the disease. Additionally, the identification of biomarkers of AD may further provide important insights into the pathogenesis of AD. To this end there have been several reports and patent applications reporting biomarkers of AD (e.g. WO 2005/052592, WO 2007/136614). However, the majority of these biomarkers have either yet to undergo full validation or have not been accepted/taken-up by the clinical community. Even if these biomarkers were to be validated and clinically utilised, there is always a need for additional biomarkers of AD with improved diagnostic and prognostic capabilities.
A prognostic test for gauging the rate of advancement of AD in patients with the disease is also clinically desirable.
Once diagnosed with AD, the average life-expectancy is from 8 to 12 years, although it can span from 3 to 20 years. The likely speed of AD progression is very much a factor of interest to the patient and patient's family, and facilitates a number of their decisions such as short and long-term care provision and participation in clinical drug trials. The progressive cognitive loss experienced by people with AD is often documented using the MMSE. This measurement provides information on the rate of change over time and aids in gauging the effectiveness of therapeutic interventions, measuring cognitive decline, and the planning of health care. However, this method has shortcomings: it requires several measurements to be taken over time, and as the patient's condition deteriorates, the procedure becomes more difficult to implement.
Doody et al. (2001) describe a method for estimating progression rates in AD using MMSE. The method requires three pieces of data: the expected MMSE value for a patient, the initial MMSE of the patient and a physician's estimate of the duration of AD in the patient. The problem of such a method is that it is resource intensive, requiring an initial MMSE test to be conducted and in-depth information to assess the duration of AD prior to the patient's presentation at the clinic. Other methods used to predict the rate of decline in AD patients have made use of expensive, specialist equipment such as magnetic resonance imaging and voxel-based morphometry. Therefore, there is also an urgent need for a rapid, supportive method that predicts whether AD patients are suffering from ACD or SCD.
There has been intensive research over the past decade to detect protein biomarkers of AD, principally in cerebrospinal fluid (CSF). Amyloid β protein and tau protein have been the subject of much research, and high levels of these proteins in CSF represent the most promising protein biomarkers of AD. However, these biomarkers have yet to receive total clinical acceptance, and the method has the added disadvantage of requiring a painful lumbar puncture procedure executed by a skilled physician, in order to obtain a CSF sample for analysis. More recently, there has been much interest in uncovering blood-derived protein biomarkers of AD (e.g. Hye et al., Zhang et al., WO 2005/052592).
The ideal biomarker would be one that is highly specific for a disease state. The complexity of the human body and its biochemical pathways, with many proteins being multifunctional and inter-dependent, suggests that such biomarkers, if existing, are likely to be rare. Furthermore, with one million proteins estimated to make-up the human proteome, it will be difficult to isolate and identify such rare proteins, and multi-biomarker combinations for disease identification are more likely. However, biomarkers with greater disease specificity, whether used alone or in combination with other biomarkers, are preferred. Regardless of the apparent specificity of an individual protein for a specific disease state, in practice its diagnostic or prognostic use is likely to be adjunctive, that is, as a support or aid to other proteins and methods in the diagnosis or prognosis of a disease.